Tyrosine phosphate (Tyr-P) covalently attached to albumin potently induced the formation of sheep antibodies (Ab) which bound [14C]Tyr-P. Affinity purified Tyr-P Ab binding to [14C]Tyr-P was not inhibitedby tyrosine sulfate, Ser-P, and Thr-P AT 50-100 fold the concentration of Tyr-P required for about 50% occupancy. The specificity of anti Tyr-P Ab for Tyr-P Ab protected Tyr-P residues in proteins from dephosphorylationby phosphatases. (2) Anti-Tyr-P Ab quantitatively precipitated phosphotyrosyl protein when incubated with anti-sheep IgG Ab. (3) Anti-Tyr-P Ab bound to phosphotyrosyl protein electrophoretically transferred to nitrocellulose paper. Little to no cross reactivity was seen with pmol levels of authentic phosphoserine proteins. Peroxidase labeled anti-sheep IgG Ab allowed visualization of proteins containing Tyr-P residues on Western blots at a sensitivity limit of about 50% fmols. Autoradiogram intensities from 32P labebled cell proteins were comparable to immunostains only after base treatment to dephosphorylate Ser-P/Thr-P residues. Immunostains of Ehrlich ascites tumor cell proteins revealed prominent bankc of a Mr about 97,000 protein found in membranes and a Mr about 3-35,000 cytosolic protein. Immunological identification of proteins containing Tyr-P may be used to characterize certain cells, and those proteins which are targets for oncogene derived protein tyrosine kinases.